

Photoreactive	Group

aryl azides	amine
acyl azides	amide
azidoformates	carbamate
sulfonyl azides	sulfonamide
phosphoryl azides	phosphoramide
diazoalkanes	new C—C bond
diazoketones	new C—C bond and ketone
diazoacetates	new C—C bond and ester
beta-keto-alpha-diazoacetates	new C—C bond and beta-ketoester
aliphatic azo	new C—C bond
diazirines	new C—C bond
ketenes	new C—C bond
photoactivated ketones	new C—C bond and alcohol

The photoactivatable nucleic acids of the invention can be applied to any surface having carbon-hydrogen bonds, with which the photoreactive groups can react to immobilize the nucleic acids to surfaces. Examples of appropriate substrates include, but are not limited to, polypropylene, polystyrene, poly(vinyl chloride), polycarbonate, poly(methyl methacrylate), parylene and any of the numerous organosilanes used to pretreat glass or other inorganic surfaces. The photoactivatable nucleic acids can be printed onto surfaces in arrays, then photoactivated by uniform illumination to immobilize them to the surface in specific patterns. They can also be sequentially applied uniformly to the surface, then photoactivated by illumination through a series of masks to immobilize specific sequences in specific regions. Thus, multiple sequential applications of specific photoderivatized nucleic acids with multiple illuminations through different masks and careful washing to remove uncoupled photo-nucleic acids after each photocoupling step can be used to prepare arrays of immobilized nucleic acids. The photoactivatable nucleic acids can also be uniformly immobilized onto surfaces by application and photoimmobilization.[0100]

Masks are known in the art and can be provided in the form of a transparent support material selectively coated with a layer of opaque material. Generally, masks are spatially distributed barrier materials that are applied to a substrate of interest to block application of photons from a portion of the substrate surface overlaid by the barrier material. Such masks shield the underlying portion of substrate from contact with activating light. Suitable masks are discussed, for example, in U.S. Pat. No. 5,831,070 (Pease et al., Nov. 3, 1998). Portions of the opaque material can be removed, leaving opaque material in the precise pattern desired on the substrate surface. The mask is brought into close proximity with, imaged on, or brought directly into contact with the master array support surface. “Openings” in the mask correspond to areas on the substrate where it is desirable to attach a desired moiety, such as mucleic acid, micro-beads, and the like. In some embodiments, the mask can be repositioned for multiple applications. Alternatively, multiple masks may be used.[0101]

Multi-Ligand Conjugate[0102]

The invention further includes a multi-ligand conjugate containing a plurality of active (e.g., binding or polymerizable) domains. The multi-ligand conjugate comprises a core having attached thereto a first ligand binding domain, a second ligand binding domain, and a third ligand. The first and second ligand binding domains are preferably nucleic acids, while the third ligand is preferably a moiety other than a nucleic acid. Preferably, the third ligand comprises a binding ligand or a polymerizable group. Alternatively, the multi-ligand conjugate comprises a core having attached thereto a second ligand binding domain, and a third ligand. In this embodiment, the second ligand binding domain is complementary to both the address ligand and a target ligand to be detected in a sample. The multi-ligand conjugate provides a conjugate that is capable of using the template established by the master array, to produce one or more replicate assay arrays as described herein.[0103]

The core of the multi-ligand conjugate provides an area for attachment of the first and second ligand binding domains, as well as the third ligand. The core comprises a suspendable particle or a soluble molecule, as described herein. Suitable suspended particles include micro-beads. Alternatively, the core comprises a molecular moiety, that is preferably soluble. As used herein, when the core of the multi-ligand conjugate is provided in the form of a micro-bead, the micro-bead will be referred to as a “transfer micro-bead.”[0104]

In one embodiment, the core is provided in the form of a transfer micro-bead. Micro-beads useful in preparing a multi-ligand conjugate of the invention can be of the same variety as those described above with respect to the “linking micro-beads.” Preferred beads are desirably spherical, homogeneous in size, with a specific gravity greater than that of water. Also, preferred beads have a chemical composition, or at least surface, that is subject to bonding with organic reagents. For example, homogeneous silica spheres of between about 0.5 micron and about 20 microns, preferably between about 1 micron and about 10 microns, more preferably between about 1 micron and about 5 microns (μ) diameter can be reacted with an organosiloxane reagent containing a thermochemically reactive group (e.g., epoxy), in order to couple the reagent to corresponding groups of a binding ligand.[0105]

An appropriate transfer micro-bead for use in the invention includes any three dimensional structure that is capable of being bound to a first ligand binding domain, second ligand binding domain, and third ligand, as herein described. Preferably, the transfer micro-bead has a density that is greater than water, to allow the beads to be brought into proximity with the surface of the master array for hybridization between the address oligonucleotide and the first binding domain of the multi-ligand conjugate. The transfer micro-bead can be provided in any suitable size, and nano-beads as well as micro-beads are contemplated in the invention.[0106]

As contemplated in the invention, the bead can be fabricated from virtually any insoluble or solid material. For example, the bead can be fabricated from silica gel, glass, nylon, Wang resin, Merrifield resin, Sephadex, Sepharose, cellulose, magnetic beads, Dynabeads, a metal surface (e.g. steel, gold, silver, aluminum, silicon and copper), a plastic material (e.g., polyethylene, polypropylene, polyamide, polyester, polyvinylidenedifluoride (PVDF)) and the like, and combinations thereof. Examples of suitable micro-beads are described, for example, in U.S. Pat. No. 5,900,481 (Lough et al, issued May 4, 1999). Other examples of suitable micro-beads are available, for example, from Interfacial Dynamics Corporation (Portland, Oreg.).[0107]

In another embodiment, the core is provided in the form of a molecular core. Preferably, the molecular core is soluble. Suitable molecular cores include multifunctional polymers or polyfunctional monomeric molecules. Examples of multifunctional polymers include linear or branched polymers, or dendrimers. Examples of suitable polyfunctional monomeric molecules include cyanuric chloride, homocysteine, cysteine, lysine, aspartic acid, glutamic acid, serine, and the like.[0108]

In one embodiment, the multi-ligand conjugate of the invention further includes a first ligand binding domain. In a preferred embodiment, the first ligand binding domain comprises a nucleic acid, preferably an oligonucleotide. Oligonucleotides are selected for the first binding domain by preparing the complementary sequence to the random sequences comprising the address ligands of the master array. Once the sequences of the address ligands are known, the sequence of the first ligand binding domain is prepared by first determining the complementary nucleic acid sequence to the address oligonucleotide, using standard Watson-Crick base pairing rules, and then synthesizing the determined nucleic acid sequence. This preparation can be accomplished using any suitable methods known in the art, for example, by solid phase DNA synthesis. Once the oligonucleotides for the first binding domain have been synthesized, a terminus of each oligonucleotide can be modified to contain an alkylamine group on a spacer, when desired.[0109]

The length of the oligonucleotides is optimized for desired hybridization strength and kinetics. Usually, the length of the first ligand binding domain is in the 20-50 nucleotide range. Preferably, the length of the first ligand binding domain is selected based upon the length of the address ligand on the master array. In a preferred embodiment, the sequences of the first binding domain are not complementary either to one another or to any known natural gene sequence and/or gene fragment with significant probability of being present in the sample to be tested. As a result, the first binding domain oligonucleotide sequences will hybridize only with their respective complementary address oligonucleotide sequences immobilized on the master array support surface. This avoids cross-reactivity (for example, cross-hybridization) between the first ligand binding domain, which is responsible for placing the multi-ligand conjugate at a spatially-defined location on the assay array support surface, and the target nucleic acid sequences screened in the test sample.[0110]

In a preferred embodiment, the second ligand binding domain of the multi-ligand conjugate also comprises a nucleic acid, preferably an oligonucleotide. Oligonucleotides are selected for the second binding domain by first selecting the target ligands (e.g., genes and/or gene fragments) to be detected by the assay array to be fabricated in accordance with the invention. The sequences of these genes and/or gene fragments to be detected may be known, or may be determined using techniques well known in the art (e.g., Sanger dideoxy method, PCR sequencing, etc.). Once the sequences of the target nucleic acid is known, the sequence of the second ligand binding domain is prepared by first determining the complementary nucleic acid sequence to the target nucleic acid, using standard Watson-Crick base pairing rules, and then synthesizing the determined nucleic acid sequence. This preparation can be accomplished using any suitable methods known in the art, for example, by solid phase DNA synthesis. Once the second binding domains have been chosen, a terminus of each oligonucleotide can be modified to contain an alkylamine group on a spacer, when desired.[0111]

In an alternative embodiment, the second ligand binding domain is selected to be complementary to an address ligand of the master array, as well as a target ligand to be detected in a sample. In this embodiment, the sequence of the second ligand binding domain is determined by selecting the target ligands (e.g., genes and/or gene fragments) to be detected by the assay array to be fabricated in accordance with the invention. The sequence of the second ligand binding domain is prepared by determining the complementary nucleic acid sequence to the target nucleic acid, using Watson-Crick base pairing rules, and then synthesizing the determined nucleic acid sequence, as discussed above. The address ligands in this embodiment will then be fabricated to include sequence that is substantially complementary to the target ligand. According to this embodiment, there is no need to provide the multi-ligand conjugate with a first ligand binding domain, since the second ligand binding domain will hybridize with the address ligands of the master array.[0113]

The multi-ligand conjugates can be fabricated to include oligonucleotides that are complementary to, and thus will hybridize with, any desired target nucleic acid found in a biological sample, and which has a known, or determinable, sequence. In one embodiment, the various second binding domains of the multi-ligand conjugates of the present invention can comprise a wide array of oligonucleotides that are complementary to various target nucleic acids found in a biological sample.[0114]

In an alternative embodiment, the second binding domains of the multi-ligand conjugates will be fabricated such that the resulting assay array will locate target nucleic acids of a particular family of naturally-occurring genes. One example of this particular embodiment would be a set of multi-ligand conjugates containing second binding domains complementary to the 50-60 known target sequences of cystic fibrosis. Thus, the resulting assay array would be fabricated such that it detects the presence of the sequences known or suspected to be associated with this disorder only.[0115]

The third ligand of the multi-ligand conjugate is a molecule that transfers the multi-ligand conjugate to the assay array prepared according to the invention. In one embodiment, once the multi-ligand conjugates have hybridized to the master array at the desired address locations, the third ligand allows the multi-ligand conjugate to be transferred to the assay array while maintaining the positional arrangement of nucleic acids established by the master array. Preferably, the third ligand is provided as a binding ligand or a polymerizable group.[0116]

In one embodiment, the third ligand is a binding ligand. As used herein, “binding ligand” means any ligand that is capable of binding to a binding partner. Preferably, the binding ligand is a member of a specific binding pair that is capable of binding with a complementary binding moiety. As contemplated in the invention, the binding ligand is a biological or synthetic molecule that has high affinity for another molecule or macromolecule, through covalent or non-covalent bonding. Preferably, the binding ligand contains (either by nature or by modification) a functional chemical group (such as a primary amine, sulfhydryl group, aldehyde, or the like), an epitope (antibody), a hapten or a ligand, that allows it to covalently react or non-covalently bind to a common functional group. For example, when the third ligand comprises avidin or streptavidin, the multi-ligand conjugate can be bound to a surface coated with biotin or derivatives of biotin such as imino-biotin. It will be appreciated that the binding members can be reversed, e.g., a biotin-coated bead can bind to a streptavidin-coated solid support. Other specific binding pairs contemplated for use in the invention include haptens (such as digoxigen, 2,4,-dichlorophenoxyacetic acid, trinitrophenyl) and high-affinity antibodies, hormone-receptor, enzyme-inhibitor, antibody-antigen and the like.[0117]

In one embodiment, the third ligand is provided in the form of a biotin-poly(alkyleneoxide) amine derivative. Such a derivative can be prepared, for instance, by reacting an N-oxysuccinimide ester of biotin with a poly(ethyleneoxide) diamine of approximately the same molecular length as the oligonucleotide derivatives to be placed on the same core or micro-bead.[0118]

The third ligand can be attached directly to the core or transfer micro-bead of the multi-ligand conjugate, or indirectly by the use of a spacer, in a manner that allows the ligand to be free of steric hindrance (e.g., steric hindrance caused by proximity of the ligand to the transfer micro-bead). In other words, use of a spacer to attach the third ligand will ensure that the ligand is sufficiently distant from the core or surface of the transfer micro-bead to allow it to perform its desired function, e.g., polymerize or bind with a corresponding binding partner contained on the assay array surface.[0119]

As used herein, a “spacer” for attachment of the third ligand to the core is a molecule that allows attachment of the third ligand to the core in a spatial relationship that does not interfere with the function or reactivity of the third ligand. Suitable spacers include polyethylene glycol, as well as water-soluble bi- or poly-functional monomers such as acrylamides, vinylpyrrolidones, carbohydrates, and the like. Preferably, such water-soluble bi- or poly-functional monomers comprise oligomers or polymers. In a preferred embodiment, the spacer comprises a hydrophilic spacer, for example, a polyethylene glycol spacer, of sufficient length to allow the binding domain to be free of steric hindrance.[0120]

In one embodiment, the multi-ligand conjugate of the present invention further comprises a polymerizable group. As used herein, “polymerizable group” refers to a molecule that is capable, under suitable conditions, of undergoing polymerization, a chemical reaction in which relatively simple molecules combine to form a macromolecule or a chain-like molecule. The third ligand of the multi-ligand conjugate can be provided in the form of a polymerizable group, e.g., to allow the conjugates to form into a stable film while in position on the master array.[0121]

Examples of suitable polymerizable groups include free-radical generators such as vinyl, acrylic, maleic, itaconic, unsaturated fatty acids, and other ethylenically- and acetylenically-unsaturated hydrocarbon groups. In a preferred embodiment, the polymerizable groups are adapted to undergo addition polymerization, in the presence of added monomers and other reagents (e.g., initiators), to form a stable film. Polymerization can take place via any suitable reaction, including aqueous free-radical solution polymerization, exposure to heat, high energy radiation, ultrasonic waves, ultraviolet radiation, and ionic polymerization catalysts to produce water-soluble or swellable polymers. Further, polymerization of the groups can take place via base-catalyzed hydrogen-transfer polymerization. Those skilled in the art, given the present description, will appreciate the manner in which such groups can be used to copolymerize the ligands with other monomers, and in turn their corresponding conjugates, into the form of an integral polymeric structure, e.g., a film backing, which can in turn be used as or transferred to an assay array support surface.[0122]

In this embodiment, once the address oligonucleotides of the master array have each hybridized with their respective first binding domains of the multi-ligand conjugates, the polymerizable group can be reacted, using reagents and conditions within the skill of those in the art, to allow the formation of a polymer film. The polymerizable groups are copolymerized into the film backing while substantially maintaining their respective positions determined by the master array. The film can be used as is, or optionally stabilized (e.g., gelled, laminated with another layer, or otherwise solidified) such that it is sufficiently stable (e.g., self supporting) to maintain the spatially-defined locations of each “address.” Simultaneously or thereafter, this polymeric backing can be transferred to, or otherwise incorporated into, an assay array surface in a manner that maintains the desired orientation of conjugates.[0123]

In accordance with the invention, the individual ligands of the multi-ligand conjugate can be attached to a core atom or molecule in any suitable manner and/or order, e.g., individually or together (e.g., in linear sequence and at a single location or at a plurality of locations). For example, in one embodiment, the ligands are attached to the core simultaneously, e.g., under similar reaction conditions. Optionally, the ligand serving as the third ligand is attached to the core after hybridization between the address oligonucleotide of the master array and the complementary oligonucleotide provided by the multi-ligand conjugate. In this embodiment, the third ligand is present as a separate reagent; however, attachment of the third ligand is capable of taking place under the same or similar reaction conditions as the first and second binding domains. In yet another embodiment, each of the ligands is attached to the core under separate conditions of separate reactions.[0124]

The sequence of attachment of these ligands to the core of the multi-ligand conjugate is not considered to be crucial to the present invention, and so long as the individual active (e.g., binding) domains are available for binding their specific complement, they can be attached to the core or to one another in any order. In a particularly preferred embodiment, each individual binding domain is attached to the core individually, at a plurality of locations on the surface.[0125]

In one embodiment, epoxy-glass surfaces are formed on the surface of the transfer micro-bead, which is thereafter reacted with the first and second ligand binding domains, as well as the third ligand. In this embodiment, glycidoxypropyltriethoxy siloxane is reacted with glass micro-beads under published reaction conditions (See “Grafting Rates of Amine-Functionalized Polystyrenes onto Epoxidized Silica Surfaces,”[0126]Macromolecules,33:1105-1107 (2000)), to provide epoxy-glass surfaces. The three binding domains of the present invention, each terminated with alkylamine groups on spacers, can be readily coupled with the epoxy groups on the surface of the bead.

First and second oligonucleotide binding domains can be attached to the transfer micro-bead in any suitable fashion, e.g., alkylamine-terminated nucleic acids can be synthesized and thermochemically attached. As described above, the first binding domain comprises an oligonucleotide that is complementary to the address oligonucleotide immobilized onto the master array support surface, while the second binding domain is complementary to a chosen target nucleic acid suspected or known to be in a sample to be tested. As discussed above, the sequences of the first ligand binding domain are preferably not complementary either to one another or to any known natural gene sequence and/or gene fragment with significant probability of being present in the biological sample.[0127]

In one embodiment, the first and second ligand binding domains are provided in the form of alkylamine nucleic acid derivatives, and the third ligand is provided in the form of a biotin hydrazide. The core is provided as a micro-bead that is provided with a photoreactive poly-N-oxysuccinimide (polyNOS) or epoxide surface. The alkylamine derivatives and biotin hydrazide react with the reactive surface of the core to allow attachment of the binding ligands and third ligand. Alternatively, 5′-OH oligonucleotide and allylamine oligonucleotide derivatives are used as the first and second ligand binding domains, and the third ligand is provided as biotin hydrazide (binding ligand) or allylamine (polymerizable group, for in situ formation of patterned film), with triazine trichloride as a soluble molecule core.[0128]

In an alternative embodiment, the third ligand is provided in solution, rather than attached to the core of the multi-ligand conjugate. In this embodiment, the third ligand is incorporated after hybridization of the master array address oligonucleotide with its complementary first binding domain of the multi-ligand conjugate. Therefore, in this embodiment, the third ligand is incorporated into the complex formed on the master array after attachment of the multi-ligand conjugate, and before the assay array is brought into binding proximity with the master array. Preferably, the addition of the third ligand does not require different reaction conditions from that of the first and second binding domains; rather, the addition of the third ligand can take place under the same conditions.[0129]

In a preferred embodiment, the third ligand is attached to the multi-ligand conjugate using photoreactive chemistry. In this embodiment, the surface of a micro-bead is pretreated, for example, with an organosiloxane in order to prepare the surface for reacting with photoreactive compounds. In this embodiment, the third ligand is attached to a hydrophilic spacer, which is, in turn, attached to a photoreactive group. The photoreactive group allows bond formation between the spacer (attached to the binding ligand) and the core of the multi-ligand conjugate.[0130]

The third ligand is preferably bound covalently to the core, whether the core comprises a suspendable particle or a soluble molecule. In one embodiment, the third ligand derivative includes a reactive group selected to form a covalent bond with a target group found or placed on the core of the multi-ligand conjugate. For example, the core may contain epoxy or active ester groups and the third ligand may contain amine or hydrazide groups; these two classes of reactive groups will react to form covalent bonds when placed together in aqueous, slightly alkaline conditions. When the core comprises a suspendable particle, for example, the biotin-poly(alkyleneoxide) amine derivative would be reacted with a soluble copolymer containing photoreactive groups and N-oxysuccinimide (NOS) ester groups (as would amine-containing derivatives of the other two ligands or ligand-binding domains). This multi-ligand conjugate derivative would be photochemically bound to the core particle.[0131]

In an alternative embodiment, wherein the invention includes the use of porous linking micro-beads immobilized onto the surface of the master array, and covalently bound to address oligonucleotides, the transfer micro-bead as the core of the multi-ligand conjugate is not required. In this embodiment, the porous linking micro-beads can be immobilized onto the surface of the master array, each porous micro-bead having a plurality of address oligonucleotides attached to its surface. According to this embodiment, the multi-ligand conjugates comprise a first binding domain complementary to the address oligonucleotide, a second binding domain complementary to a target ligand found in a biological sample, and a third ligand comprising a binding ligand. The multi-ligand conjugate preferably does not contain a transfer micro-bead, but rather a chemical (e.g., molecular) core.[0132]

A plurality of multi-ligand conjugates are applied to the master array and incubated under conditions suitable to allow hybridization of the address sequence of the master array with its complementary oligonucleotide contained within the multi-ligand conjugate. Those skilled in the art will be able to determine suitable conditions of temperature, salt concentration, and buffer composition for hybridization of address oligonucleotides and first binding domains. For a discussion of hybridization conditions and methods for applying them, see pages 3-15, 62-66, 73-112[0133], Nucleic Acid Hybridization: A Practical Approach, Hames and Higgins, eds. (1985), the disclosure of which is incorporated herein by reference.

Assay Array Support[0134]

The assay array support of the invention comprises a support surface that is preferably coated with binding sites for the third ligand of the multi-ligand conjugate. The assay array support provides the support for a replicate array to be fabricated using the master array.[0135]

The assay array support can be fabricated from any suitable material to provide an optimal combination of such properties as stability of dimension, shape, and smoothness. Suitable materials for use in fabricating the assay array include those described as being useful for the master array itself, and preferably include silicon and glass. In a particularly preferred embodiment, the assay support is provided with planar dimensions of between about 0.5 cm and about 7.5 cm in length, and between about 0.5 cm and about 7.5 cm, preferably between about 1 cm and about 2 cm, in width. Preferably, the assay array support is of comparable dimensions to the master array, in order to provide direct spatial relationships.[0136]

In yet another embodiment, multiple arrays can be replicated onto a single assay array support. In this embodiment, one or more master arrays are used to create individual replicate arrays in defined areas on the assay array support. The resulting assay array contains multiple probe arrays on a single support surface, and the composition of each array can be tailored for a particular use, as desired.[0137]

As used herein, a “binding site” refers to an area on the surface of the assay array that is capable of binding the third ligand of the multi-ligand conjugate. Preferably, the binding sites comprise members of a specific binding pairs that are capable of binding with a complementary binding moiety, e.g., the third ligand of the multi-ligand conjugate, in a covalent or non-covalent manner. Suitable specific binding pairs are described herein with respect to the third ligand of the multi-ligand conjugate. One of skill in the art can select suitable binding sites based upon the composition of the multi-ligand conjugate and desired use of the assay array.[0138]

The surface of a discrete assay array support can be pretreated to facilitate attachment of the oriented conjugate layer. In a preferred embodiment, the assay array is provided in the form of a silicon chip that has been cleaned, polished and treated to provide a smooth silicon oxide surface. The silicon oxide surface can then be treated with an organosiloxane reagent (as described herein) to provide a smooth photoactivatable surface.[0139]

In one embodiment, the assay array support of the present invention can be coated with binding partner molecules available to form bonds with the third ligand of the multi-ligand conjugate. The assay array support surface can be prepared by coating the surface with a photoactivatable compound (e.g., photoreactive siloxane reagent) to render the surface photoreactive. Thereafter, the assay array support surface can be exposed to a solution containing binding partner molecules (e.g., avidin), and the solution illuminated, in order to allow bond formation between the binding partner and surface of the assay array. Optionally, the surface of the assay array is passivated prior to and/or after exposure to the binding partner, e.g., with a surfactant (e.g., a biotin triblock copolymer), to prevent non-specific binding of molecules found in the test sample to the assay array support surface.[0140]

In a preferred embodiment, the binding partner is provided in the form of a monolayer upon the assay array support surface. Optionally, the binding partner can be provided in the form of a three-dimensional layer, e.g., an avidin-hydrogel composite of sufficient thickness, for example, up to about 20 microns (μ) thickness. Thickness of the three-dimensional layer will depend upon such factors as the molecular weight of the polymer used, the swellability of the polymer (e.g., hydrophilicity), and the quantity (e.g., concentration and volume) of material applied. Suitable thickness is about 0.1μ to about 20μ, preferably about 0.5μ to about 10μ, more preferably about 0.5μ to about 5μ. Such a structure can be obtained, for instance, by loading the photoreactive assay array surface with a solution of avidin plus photoreactive hydrogel (e.g., copolymer of benzoylbenzamidopropyl methacrylamide with vinylpyrollidone or acrylamide) and illuminating while wet.[0141]

In another preferred embodiment, the linking partner is provided by a thin film of photopolymer that is applied to a dimensionally stable assay array support surface. A self-assembling monolayer film of passivating surfactant containing a high surface density (e.g., 0.1 pmol/mm[0142]²) of high-affinity ligand group (e.g., biotin) is applied to the photoreactive surface. The monolayer surfactant film is then photocoupled to the surface, and any unbound surfactant is rinsed away. The passivated, ligand-loaded surface is saturated with high-affinity binding partner molecule (e.g., x-avidin), and any excess binding partner is rinsed away. Suitable high-affinity binding partner molecules include, for example, x-avidin film, or high-affinity antibody (e.g., anti-digoxygenin).

Optionally, the system further includes other components as well, including for instance, reagents or other mechanisms for use in disassociating complementary, hybridized oligonucleotides (e.g., the address oligonucleotide of the master array and its complementary oligonucleotide carried by the multi-ligand conjugate). For example, suitable mechanisms for disassociating the hybridized DNA include techniques or reagents that alter the temperature, pH, or salt concentration of the system. One of skill in the art could apply such mechanisms to dissociate hybridized DNA and achieve the invention herein.[0143]

Optionally, the system further comprises a scanner, as commercially available (e.g., a confocal fluorescence microscope as available from Molecular Dynamics) to detect the hybridization of nucleic acid targets to specific addresses.[0144]

Method[0145]

In another aspect, the invention provides a method for reproducibly preparing a nucleic acid array, the method comprising:[0146]

a) providing a master array having a support surface;[0147]

b) immobilizing a plurality of address ligands on the master array support surface in the form of a patterned array;[0148]

c) determining the pattern of the immobilized address oligonucleotides;[0149]

d) providing a plurality of multi-ligand conjugates, each multi-ligand conjugate comprising: (i) a core; (ii) at least one molecule of a first ligand binding domain, comprising a ligand selected to bind in a complementary manner to an address ligand of the master array, (iii) at least one molecule of a second ligand binding domain, comprising a ligand selected to bind in a complementary manner to a particular target ligand, and (iv) at least one molecule of a third ligand, wherein the first ligand binding domain, second ligand binding domain, and third ligand are attached to the core;[0150]

e) contacting and incubating the multi-ligand conjugates with the master array support surface under conditions suitable to allow each address ligand to bind its complementary first ligand binding domain of the multi-ligand conjugates;[0151]

f) providing an assay array support surface,[0152]

g) contacting the bound multi-ligand conjugates with the assay support surface under conditions suitable to permit the conjugates to be transferred to the assay support in a pattern corresponding to their pattern on the master array; and[0153]

h) disassociating the first ligand binding domain from the master array support in a manner that permits the conjugates to remain upon the assay support surface in the desired pattern.[0154]

In another aspect, the invention provides a method for reproducibly preparing a nucleic acid array comprising the steps of:[0155]

a) providing a master array having a support surface;[0156]

b) immobilizing a plurality of address ligands on the master array support surface in the form of a patterned array;[0157]

c) determining the pattern of the immobilized address oligonucleotides;[0158]

d) providing a plurality of multi-ligand conjugates, each multi-ligand conjugate comprising: (i) a core; (ii) at least one molecule of a second ligand binding domain, comprising a ligand selected to bind in a complementary manner to a particular address ligand and to a particular target ligand, and (iii) at least one molecule of a third ligand, wherein the second ligand binding domain and the third ligand are attached to the core;[0159]

e) contacting and incubating the multi-ligand conjugates with the master array support surface under conditions suitable to allow each address ligand to bind its complementary second ligand binding domain of the multi-ligand conjugates;[0160]

f) providing an assay array support surface,[0161]

g) contacting the bound multi-ligand conjugates with the assay support surface under conditions suitable to permit the conjugates to be transferred to the assay support in a pattern corresponding to their pattern on the master array; and[0162]

h) disassociating the second ligand binding domain from the master array support in a manner that permits the conjugates to remain upon the assay support surface in the desired pattern.[0163]

Optionally, the method further comprises the step of using the assay array support surface fabricated in step h) to prepare a second assay array. A second set of multi-ligand conjugates are used to generate a second assay array that is a substantially identical copy of the original master array. The second set of multi-ligand conjugates can be fabricated according to the teachings herein. In this embodiment, the nucleic acid sequences of the second assay array are substantially identical to the nucleic acid sequences of the original master array.[0164]

In a preferred embodiment, immobilization of the address ligands onto the master array support surface is random, and the location of each address is determined after the address ligands are immobilized on the surface. Preferably, the address ligand, first ligand binding domain, and second ligand binding domain are nucleic acid sequences, and the third ligand comprises a binding ligand or a polymerizable group. In use, the core of the multi-ligand conjugate bearing three or more ligands can be mixed in equal numbers and suspended in aqueous buffer. This suspension can contain an excess (over the hybridization capacity of the micro-beads on the master array) of each transfer micro-bead carrying the oligonucleotide sequence complementary to a characteristic oligonucleotide sequence of a target nucleic acid expected to be detected in a sample.[0165]

The master array surface can be flooded with this suspension of the chosen mixture of multi-ligand conjugates and solid-state hybridization can be allowed to proceed to near equilibrium. The excess multi-ligand conjugates can then be recovered from the hybridized master array by rinsing, and the bound conjugates either copolymerized to form a stable backing (when the third ligand comprises a polymerizable group) or bound to corresponding binding partners (when the third ligand comprises a binding ligand) on the assay array. The master array is then available for a repeat replication process.[0166]

The master array can be used repeatedly to prepare corresponding assay arrays, which in turn can be packaged, stored and transported, e.g., in the wet or dry state. Upon removal from its package, an assay array will be ready to use in a routine hybridization assay protocol with a scanner. Moreover, the present invention provides a master array that is versatile to use—the master array can be used and reused, to create substantially identical arrays or to change the function of the array (e.g., to alter the oligonucleotide sequences complementary to a characteristic oligonucleotide sequences of target nucleic acid, such that the user can alter the target nucleic acid to be detected in a sample).[0167]

Those skilled in the art, given the present description, will appreciate the manner in which commercially available microarrays can themselves be used as master arrays of the present invention, to be replicated one or more times to form corresponding assay arrays. In this case, the individual nucleic acids of the commercial array, regardless of their original specificity, can each be considered as mere address sequences, and used in the manner described herein to provide assay arrays of any desired specificity. The user can determine the sequences of each address and fabricate the first binding ligand of the multi-ligand conjugate using the teachings herein.[0168]

In yet a further aspect, and particularly where the third ligand is provided in the form of a binding ligand, the present invention provides a sandwich array comprising:[0169]

a) a master array comprising a support surface having a plurality of address ligands immobilized thereon;[0170]

b) a plurality of multi-ligand conjugates, each multi-ligand conjugate comprising a core having attached thereto; (i) molecules of a first ligand binding domain, each molecule comprising a ligand selected to bind in a complementary manner to a particular address ligand of the master array, (ii) molecules of a second ligand binding domain, each molecule comprising ligand selected to bind in a complementary manner to a characteristic target ligand; and (iii) at least one molecule of a third ligand, wherein the first ligand binding domain is bound to the corresponding address ligand immobilized onto the master array; and[0171]

c) an assay array support comprising a support surface coated with attachment sites for the third ligand, wherein the attachment site of the assay array is bound to the corresponding binding ligand of the multi-ligand conjugates.[0172]

Optionally, the sandwich array further comprises a linking micro-bead immobilized onto the surface of the master array, the linking micro-bead having attached thereto a plurality of address oligonucleotides.[0173]

Kit[0174]

In yet another alternative embodiment, the present invention provides a corresponding kit comprising one or more components for reproducibly preparing a nucleic acid array, the kit components selected from the group consisting of:[0175]

a) a master array comprising a support surface having a plurality of “address” ligands immobilized thereon, the ligands being immobilized in the form of a patterned array;[0176]

b) a plurality of multi-ligand conjugates, each multi-ligand conjugate comprising a core having attached thereto: (i) molecules of a first ligand binding domain, each molecule comprising a ligand selected to bind in a complementary manner to a particular address ligand of the master array, (ii) molecules of a second ligand binding domain, each molecule comprising a ligand selected to bind in a complementary manner to a characteristic target ligand, and (iii) at least one molecule of a third ligand;[0177]

c) an assay array support comprising a support surface for the replicate array coated with attachment sites for the third ligand.[0178]

Optionally, the kit of the invention further comprises reagents for performing the assay described herein, such as suitable buffers to provide appropriate pH levels, salt concentrations, and the like to perform hybridization. The kit can further comprise an array reader for determining the location of address oligonucleotides on the master array and the presence of hybridized target nucleic acids on the assay array.[0179]

Reusable Nucleic Acid Array[0180]

In yet another alternative embodiment, the present invention provides a method and system for directly preparing a replicable array in the form of a reusable nucleic acid array. In this embodiment, the surface to which the nucleic acid is immobilized is provided by an optical fiber array, the master array support surface being provided by the distal ends of the fibers themselves. Optionally, and preferably, micro-wells are etched into the distal ends of the optical fibers.[0181]

In such an embodiment, a conventional optical fiber bundle can be adapted for use as a specific binding array biosensor system for detecting a variety of target nucleic acids in a sample. Commercial sources provide optical imaging fiber bundles comprising thousands (e.g., 3,000-100,000) of hexagonally packed, individually-coated optical fibers. Each fiber in the array carries its own, isolated optical signal from one end of the fiber to the other. Individual specific binding ligands can be bound to one end of each fiber and the other end is functionally connected to a optical signal analyzer (e.g., a CCD camera).[0182]

The assay end (containing specific-binding ligands) of the fiber bundle is exposed to a sample solution containing the unknown target and other necessary reagents (if any) of the optical assay system. The optical signal pattern is recorded and analyzed at the other end of the bundle, using the image analyzer system.[0183]

Applicant has found that oligonucleotide binding domains, complementary to target sequences, can be attached to the optical assay array support surface in any suitable manner, e.g., either directly or by means of reversibly or irreversibly bound linking molecules or micro-beads. In turn, the ultimate location of the oligonucleotide binding domains can be determined (e.g., by optical means and assay), preferably controlled (e.g., by the use of reusable address domains), and optionally replicated (e.g., by the use of reversible linkers) onto flat surface.[0184]

In one preferred embodiment, the system comprises a plurality of multi-ligand conjugates, each multi-ligand conjugate comprising molecules of a) a first oligonucleotide binding domain adapted to bind with a corresponding address ligand, and b) a second oligonucleotide binding domain complementary to a characteristic oligonucleotide sequence of a target nucleic acid (e.g., a gene and/or gene fragment). The address ligands are immobilized onto the distal end of the master array, either directly, or by the use of a linker or micro-bead. The multi-ligand conjugates would then load each of the addressed fibers with a known analyte-specific ligand. The set of analyte-specific ligands on the fibers in the bundle can be changed at will.[0185]

In another embodiment, attachment of the oligonucleotide binding domain is accomplished using a micro-bead. In this embodiment, the micro-bead is prepared such that it is attached to the oligonucleotide binding domain. The micro-bead and binding domain are then attached to the assay array support surface. Attachment of the micro-bead can be accomplished using any suitable binding pair, such as, for example, avidin-biotin. In an alternative embodiment, photoreactive reagents can be used to attach the micro-bead to the assay array support surface. Attachment of the address oligonucleotide to the micro-bead, as well as attachment of the micro-bead to the surface of the master array, can be accomplished using any suitable method herein described.[0186]

Optionally, the system further comprises an image analyzer. In this embodiment, the bundle of fibers is connected at one end to an optical sensor that collects signals and processes information from these multiple sensors (e.g., fibers).[0187]

In such an approach the invention provides a reusable nucleic acid array, in which the user is able to change the function of the nucleic acid array for each particular use. For example, the linker or micro-bead can be coated with iminobiotin, and the master array support surface can be coated with biotin. The linker or micro-bead can thus be reversibly attached to the assay array support surface. The linker or micro-bead can be dissociated by altering the pH such that the lower pH causes the linker or micro-bead to dissociate with the assay array support surface.[0188]

In one embodiment, micro-wells can be etched into the distal end of each optical fiber using techniques known in the art. For example, a wet chemical etching procedure can be used to selectively etch the cores of the individual fibers. This technique takes advantage of the difference in etch rates between the core and cladding materials of the fiber. By controlling the etching time, those of skill in the art will appreciate the manner in which high-density, ordered micro-well arrays of known shape and well volume are obtained. Well architecture is determined by the preformed imagining fiber. Since each micro-well is contained at the end of its own optical fiber, each well can serve as an individual sensor. Preferably, the diameter of the etched micro-wells is slightly larger than that of individual linking micro-beads used in connection therewith.[0189]

In one embodiment, for instance, a solution containing a plurality of micro-beads having one or more oligonucleotide binding domains attached thereto is added to the surface of a bundle of optical fibers. Preferably, the oligonucletode binding domains are complementary to a target nucleic acid to be detected and/or quantitated. The individual linking micro-beads randomly settle into each micro-well as the solution is allowed to evaporate from the optical fiber bundle surface. Optionally, and in the event a photoreactive compound is used in connection with this embodiment, the optical fibers are illuminated, to allow the linking micro-bead to photocouple to the surface of each optical fiber. Optionally, excess micro-beads are then washed from the surface of the fiber bundle.[0190]

For instance, immobilization of a bead or oligonucleotide binding domain onto the distal end of the fiber, within the micro-well, can be accomplished using photoactivatable compounds to form bonds with the well surface. In one embodiment, the well surface of the optical fiber is coated with a photoreactive siloxane reagent, and thereafter, a solution containing free (i.e., unbound) oligonucleotide binding domain is applied to the well surface. The solution is then illuminated, activating the photoreactive groups and causing bond formation between the oligonucleotide binding domain and well surface. Illumination is accomplished by passing activating light through the fiber into the well.[0191]

As discussed above, each individual fiber of the fiber bundle can be etched at one end to provide a concave receptacle for a micro-bead. A photoreactive organosiloxane reagent can be prepared by bonding a commercial organosiloxane reagent, such as aminopropyldimethyl methoxy siloxane, with a thermochemically reactive photoreagent, such as the acid chloride of benzoylbenzoic acid. The photoreactive siloxane reagent is reacted with the concave silicon oxide surface of the etched optical fibers in the image analyzer bundle. In an alternative embodiment, each micro-well is coated with x-avidin to create a surface that will react with biotin. In this embodiment, the micro-beads are coated with biotin or iminobiotin derivatives. Any specific binding pair, as described herein, will be suitable for such attachment.[0192]

Preferably, the linking micro-beads are spherical, and of a diameter approximately equal to (or slightly less than) that of the individual optical fibers. The linking micro-beads of the present invention are fabricated from any suitable material, including, for example, glass, polystyrene, polyvinylbenzophenone, photopolysiloxane-coated glass. Preferably, the micro-beads have a specific gravity greater than that of water, and have a chemical composition that is subject to bonding with organic reagents.[0193]

The oligonucleotide derivatives can be synthesized according to published and commercially utilized methods, as described in more detail herein. The length of the oligonucleotide sequences are optimized for desired hybridization strength and speed (usually in the 20-50 nucleotide range). The oligonucleotide sequences are complementary to the chosen target nucleic acid.[0194]

The “address oligonucleotide” derivatives can be coupled to the epoxy-activated linking micro-beads for the optical fiber ends. In a preferred embodiment, for example, homogeneous silica spheres of approximately the fiber diameter are reacted with a commercial organosiloxane reagent containing a thermochemically reactive group (e.g., epoxy) useful for coupling to amine groups of the oligonucleotide derivatives in this invention. Glycidoxypropyltriethoxy siloxane is reacted with the glass micro-beads under published reaction conditions, to provide an epoxy-glass surface. The address oligonucleotide derivatives of this invention terminate with alkylamine groups on spacers to couple readily with the epoxy groups on the surface of the linking micro-beads.[0195]

The bundle of etched photoreactive optical fibers is exposed to a slurry of linking micro-beads (in sufficient number to provide at least one type of address oligonucleotide per fiber), each micro-bead having address oligonucleotide sequences immobilized on its surface. Preferably, one oligonucleotide binding domain is bound to each optical fiber well. In this embodiment, the master array is reusable. This slurry is comprised of approximately equal numbers of beads with each of the desired address oligonucleotide sequences. The number of beads providing oligonucleotide sequences is preferably sufficient to provide a redundancy (at least 3-fold) of each target nucleic acid specific site. As the solvent (preferably aqueous) evaporates from the slurry pool on the photoreactive surface in the dark or dim light, the uniform-sized beads are left in the concave pockets at the ends of the ordered arrangement of fibers. The linking micro-beads providing oligonucleotide sequences are then “fixed” by illumination in the dry state to form carbon-carbon bonds between the photoreactive groups on the optical fiber surface and the organic groups (mostly oligonucleotides) on the address oligonucleotide beads. Excess micro-beads are rinsed from the system.[0196]

The resultant optical fiber bundle will have a stable and ordered arrangement of the oligonucleotides complementary to target nucleic acid sequences on one end of its fibers, and the locations of the oligonucleotide sequences can be then determined by conventional methods of fluorescent hybridization, comprising sequential addition of fluorescent-labeled, complementary sequences to the array, with rinsing and array reading between each addition, as discussed in detail herein.[0197]

In one preferred embodiment, complementary sequences are labeled with fluorophores. Fluorescent-labeled oligonucleotides complementary to each of the sequences are synthesized, each containing several (e.g., one of ten) different fluorescent compounds that can be distinguished from each other. Each oligonucleotide, therefore, has a known sequence and a known identifiable label. The several oligonucleotides, each having a different, known sequence complementary to an oligonucleotide sequence on the surface and one of a comparable number of distinguishable fluorophores, are simultaneously applied to the surface and hybridized at 55° C. for 30 minutes.[0198]

The surface is then rinsed and analyzed to identify the locations of each of the sequences. Another set of sequences is then hybridized to the surface and scanned to identify the second set of sequences. This process is continued until all of the locations are identified, after which the master array is stripped by immersing in boiling water for two minutes. It is then ready for use to detect the presence of target nucleic acids in a sample.[0199]

In use, the master array is exposed to a test solution containing a plurality of target nucleic acid sequences (e.g., genes and/or gene fragments). The test solution is labeled via a fluorescent label to permit visualization of the results. The test solution is incubated under conditions to allow the oligonucleotide binding domain to hybridize to the target nucleic acid contained within the sample. Unbound solution is then rinsed from the master array. The hybridized nucleic acid sequences are visualized using the CCD camera of the array system described above.[0200]

The assay arrays are washed with phosphate buffered saline containing 0.05% Tween 20 (PBS/Tween), then blocked with hybridization buffer, which consists of 4× SCC (0.6 M NaCl, 0.06 M citrate, pH 7.0), 0.1% (w/v) lauroylsarcosine, and 0.02% (w/v) sodium dodecyl sulfate, at 55° C. for 30 minutes. A sample containing DNA having a sequence complementary to one of the immobilized oligonucleotide probes on the array is applied to the support surface of the fiber and incubated for one hour at 55° C. in a sealed hybridization chamber. The support surface is then washed with 2× SSC containing 0.1% SDS (sodium dodecylsulfate) for 5 minutes at 55° C., after which 50 fmole of a fluorescent-labeled detection probe that is complementary to another sequence on the target DNA is applied to the support surface and incubated for one hour at 55° C. The support surface is then washed with 2× SSC containing 0.1% SDS for 5 minutes at 55° C., followed by a wash with 0.1× SSC, then dried and the fluorescence pattern recorded by the image analysis instrument.[0201]

One of skill in the art will appreciate that the present invention can provide a variety of oligonucleotide sequences in association with a particular fiber bundle. In one embodiment, for example, the fiber wells are coated individually with specific binding agents. In another embodiment, the fiber wells are coated simultaneously with a general binding agent (e.g., iminobiotin). In this embodiment, the fiber wells are loaded from a mixture of specific binding agent micro-beads each containing the complement to the general binding agent (e.g., x-avidin).[0202]

Optionally, the system further includes an image analyzer system, e.g., a CCD camera with associated software. The resultant assay array can be used in a conventional manner, e.g., the assay array can be read by a standard array reader, while the master array can be re-used to prepare more assay arrays by repeating some or all of the steps set forth above.[0203]

It will be apparent to one of skill in the art that the invention can be applied to any molecular species involved in molecular recognition. While the discussion has provided details regarding nucleic acid applications of the invention, it is understood that the invention is not so limited.[0204]

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.[0205]

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.[0206]

EXAMPLESExample 1Preparation of 4-Benzoylbenzoyl Chloride (BBA-Cl) (Compound I)

4-Benzoylbenzoic acid (BBA), 1.0 kg (4.42 moles), was added to a dry 5 liter Morton flask equipped with reflux condenser and overhead stirrer, followed by the addition of 645 ml (8.84 moles) of thionyl chloride and 725 ml of toluene. Dimethylformamide, 3.5 ml, was then added and the mixture was heated at reflux for four (4) hours. After cooling, the solvents were removed under reduced pressure and the residual thionyl chloride was removed by three evaporations using 3×500 ml of toluene. The product was recrystallized from 1:4 toluene:hexane to give 988 g (91% yield) after drying in a vacuum oven. Product melting point was 92-94° C. Nuclear magnetic resonance (NMR) analysis at 80 MHz ([0207]¹H NMR (CDCl₃)) was consistent with the desired product: aromatic protons 7.20-8.25 (m, 9H). All chemical shift values are in ppm downfield from a tetramethylsilane internal standard. The final compound was stored for use in the preparation of a monomer used in the synthesis of photoactivatable polymers as described, for instance, in Example 3.

Example 2Preparation of N-(3-Aminopropyl)methacrylamide Hydrochloride (APMA) (Compound II)

A solution of 1,3 diaminopropane, 1910 g (25.77 moles), in 1000 ml of CH[0208]₂Cl₂was added to a 12 liter Morton flask and cooled on an ice bath. A solution of t-butyl phenyl carbonate, 1000 g (5.15 moles), 250 ml of CH₂Cl₂was then added dropwise at a rate which kept the reaction temperature below 15° C. Following the addition, the mixture was warmed to room temperature and stirred two (2) hours. The reaction mixture was diluted with 900 ml of CH₂Cl₂and 500 g of ice, followed by the slow addition of 2500 ml of 2.2 N NaOH. After testing to insure the solution was basic, the product was transferred to a separatory funnel and the organic layer was removed and set aside as extract #1. The aqueous fraction was then extracted with 3× 1250 ml of CH₂Cl₂, keeping each extraction as a separate fraction. The four organic extracts were then washed successively with a single 1250 ml portion of 0.6 N NaOH beginning with fraction #1 and proceeding through fraction #4. This wash procedure was repeated a second time with a fresh 1250 ml portion of 0.6 N NaOH. The organic extracts were then combined and dried over Na₂SO₄. Filtration and evaporation of solvent to a constant weight gave 825 g of N-mono-t-butoxycarbonyl- 1,3-diaminopropane which was used without further purification.

A solution of methacrylic anhydride, 806 g (5.23 moles), in 1020 ml of CHCl[0209]₃was placed in a 12 liter Morton flask equipped with overhead stirrer and cooled on an ice bath. Phenothiazine, 60 mg, was added as an inhibitor, followed by the dropwise addition of N-mono-t-butoxycarbonyl-1,3-diaminopropane, 825 g (4.73 moles), in 825 ml of CHCl₃. The rate of addition was controlled to keep the reaction temperature below 10° C. at all times. After the addition was complete, the ice bath was removed and the mixture was left to stir overnight. The product was diluted with 2400 ml of water and transferred to a separatory funnel. After thorough mixing, the aqueous layer was removed and the organic layer was washed-with 2400 ml of 2 N NaOH, insuring that the aqueous layer was basic. The organic layer was then dried over Na₂SO₄and filtered to remove drying agent. A portion of the CHCl₃solvent was removed under reduced pressure until the combined weight of the product and solvent was approximately 3000 g. The desired product was then precipitated by slow addition of 11.0 liters of hexane to-the stirred CHCl₃solution, followed by overnight storage at 4° C. The product was isolated by filtration and the solid was rinsed twice with a solvent combination of 900 ml of hexane and 150 ml of CHCl₃.

Thorough drying of the solid gave 900 g of N-[N′-(t-butyloxycarbonyl)-3-aminopropyl]-methacrylamide, melting point 85.8° C. by DSC. Analysis on an NMR spectrometer was consistent with the desired product:[0210]¹H NMR (CDCl₃) amide NH's 6.30-6.80, 4.55-5.10 (m, 2H), vinyl protons 5.65, 5.20 (m, 2H), methylenes adjacent to N 2.90-3.45 (m, 4H), methyl 1.95 (m, 3H), remaining methylene 1.50-1.90 (m, 2H), and t-butyl 1.40 (s, 9H).

A 3-neck, 2 liter round bottom flask was equipped with an overhead stirrer and gas sparge tube. Methanol, 700 ml, was added to the flask and cooled on an ice bath. While stirring, HCl gas was bubbled into the solvent at a rate of approximately 5 liters/minute for a total of 40 minutes. The molarity of the final HCl/MeOH solution was determined to be 8.5 M by titration with 1 N NaOH using phenolphthalein as an indicator. The N-[N′-(t-butyloxycarbonyl)-3-aminopropyl]methacrylamide, 900 g (3.71 moles), was added to a 5 liter Morton flask equipped with an overhead stirrer and gas outlet adapter, followed by the addition of 1150 ml of methanol solvent. Some solids remained in the flask with this solvent volume. Phenothiazine, 30 mg, was added as an inhibitor, followed by the addition of 655 ml (5.57 moles) of the 8.5 M HCl/MeOH solution. The solids slowly dissolved with the evolution of gas but the reaction was not exothermic. The mixture was stirred overnight at room temperature to insure complete reaction. Any solids were then removed by filtration and an additional 30 mg of phenothiazine were added. The solvent was then stripped under reduced pressure and the resulting solid residue was azeotroped with 3×1000 ml of isopropanol with evaporation under reduced pressure. Finally, the product was dissolved in 2000 ml of refluxing isopropanol and 4000 ml of ethyl acetate were added slowly with stirring. The mixture was allowed to cool slowly and was stored at 4° C. overnight. Compound II was isolated by filtration and was dried to constant weight, giving a yield of 630 g with a melting point of 124.7° C. by DSC. Analysis on an NMR spectrometer was consistent with the desired product:[0211]¹H NMR (D₂O) vinyl protons 5.60, 5.30 (m, 2H), methylene adjacent to amide N 3.30 (t, 2H), methylene adjacent to amine N 2.95 (t, 2H), methyl 1.90 (m, 3H), and remaining methylene 1.65-2.10 (m, 2H). The final compound was stored for use in the preparation of a monomer used in the synthesis of photoactivatable polymers as described, for instance, in Example 3.

Example 3Preparation of N-[3-(4-Benzoylbenzamido)propyl]methacrylamide (BBA-AMPA) (Compound III)

Compound II 120 g (0.672 moles), prepared according to the general method described in Example 2, was added to a dry 2 liter, three-neck round bottom flask equipped with an overhead stirrer. Phenothiazine, 23-25 mg, was added as an inhibitor, followed by 800 ml of chloroform. The suspension was cooled below 10° C. on an ice bath and 172.5 g (0.705 moles) of Compound I, prepared according to the general method described in Example 1, were added as a solid. Triethylamine, 207 ml (1.485 moles), in 50 ml of chloroform was then added dropwise over a 1-1.5 hour time period. The ice bath was removed and stirring at ambient temperature was continued for 2.5 hours. The product was then washed with 600 ml of 0.3 N HCl and 2×300 ml of 0.07 N HCl. After drying over sodium sulfate, the chloroform was removed under reduced pressure and the product was recrystallized twice from 4:1 toluene:chloroform using 23-25 mg of phenothiazine in each recrystallization to prevent polymerization. Typical yields of Compound III were 90% with a melting point of 147-151 ° C. Analysis on an NMR spectrometer was consistent with the desired product:[0212]¹H NMR (CDCl₃) aromatic protons 7.20-7.95 (m, 9H), amide NH 6.55 (broad t, 1H), vinyl protons 5.65, 5.25 (m, 2H), methylene adjacent to amide N's 3.20-3.60 (m, 4H), methyl 1.95 (s, 3H), and remaining methylene 1.50-2.00 (m, 2H). The final compound was stored for use in the synthesis of photoactivatable polymers as described, for instance, in Example 6.

Example 4Preparation of N-Succinimidyl 6-Maleimidohexanoate (MAL-EAC-NOS) (Compound IV)

A functionalized monomer was prepared in the following manner, and was used as described in Example 6 to introduce activated ester groups on the backbone of a polymer. 6-Aminohexanoic acid, 100 g (0.762 moles), was dissolved in 300 ml of acetic acid in a three-neck, 3 liter flask equipped with an overhead stirrer and drying tube. Maleic anhydride, 78.5 g (0.801 moles), was dissoved in 200 ml of acetic acid and added to the 6-aminohexanoic acid solution. The mixture was stirred one hour while heating on a boiling water bath, resulting in the formation of a white solid. After cooling overnight at room temperature, the solid was collected by filtration and rinsed with 2×50 ml of hexane. After drying, the typical yield of the (Z)-4-oxo-5-aza-2-undecendioic acid was 158-165 g (90-95%) with a melting point of 160-165° C. Analysis on an NMR spectrometer was consistent with the desired product:[0213]¹H NMR (DMSO-d₆) amide proton 8.65-9.05 (m, 1H), vinyl protons 6.10, 6.30 (d, 2H), methylene adjacent to nitrogen 2.85-3.25 (m, 2H), methylene adjacent to carbonyl 2.15 (t, 2H), and remaining methylenes 1.00-1.75 (m, 6H).

(Z)-4-Oxo-5-aza-2-undecendioic acid, 150.0 g (0.654 moles), acetic anhydride, 68 ml (73.5 g, 0.721 moles), and phenothiazine, 500 mg, were added to a 2 liter three-neck round bottom flask equipped with an overhead stirrer. Triethylamine, 91 ml (0.653 moes), and 600 ml of tetrahydrofuran (THF) were added and the mixture was heated to reflux while stirring. After a total of four (4) hours of reflux, the dark mixture was cooled to less than 60° C. and poured into a solution of 250 ml of 12 N HCl in 3 liters of water. The mixture was stirred three (3) hours at room temperature and then was filtered through a filtration pad (Celite 545, J. T. Baker, Jackson, Tenn.) to remove solids. The filtrate was extracted with 4×500 ml of chloroform and the combined extracts were dried over sodium sulfate. After adding 15 mg of phenothiazine to prevent polymerization, the solvent was removed under reduced pressure. The 6-maleimidohexanoic acid was recrystallized from 2:1 hexane:chloroform to give typical yields of 76-83 (55-60%) with a melting point of 81-85° C. Analysis on an NMR spectrometer was consistent with the desired product:[0214]¹H NMR (CDCl₃) maleimide protons 6.55 (s, 2H), methylene adjacent to nitrogen 3.40 (t, 2H), methylene adjacent to carbonyl 2.30 (t, 2H), and remaining methylenes 1.05-1.85 (m, 6H).

The 6-maleimidohexanoic acid, 20.0 g (94.7 mmol), was dissolved in 100 ml of chloroform under an argon atmosphere, followed by the addition of 41 ml (0.47 mol) of oxalyl chloride. After stirring for 2 hours at room temperature, the solvent was removed under reduced pressure with 4×25 ml of additional chloroform used to remove the last of the excess oxalyl chloride. The acid chloride was dissolved in 100 ml of chloroform, followed by the addition of 12 g (0.104 mol) of N-hydroxysuccinimide and 16 ml (0.114 mol) of triethylamine. After stirring overnight at room temperature, the product was washed with 4×100 ml of water and dried over sodium sulfate. Removal of solvent gave 24 g of product (82%) which was used without further purification. Analysis on an NMR spectrometer was consistent with the desired product:[0215]¹H NMR (CDCl₃) maleimide protons 6.60 (s, 2H), methylene adjacent to nitrogen 3.45 (t, 2H), succinimidyl protons 2.80 (s, 4H), methylene adjacent to carbonyl 2.55 (t, 2H), and remaining methylenes 1.15-2.00 (m, 6H). The final compound was stored for use in the synthesis of photoactivatable polymers as described, for instance, in Example 6.

Example 5Preparation of Functionalized Monomer: N-Succinimidyl 6-Methacrylamidohexanoate (MA-EAC-NOS) (Compound V)

A functionalized monomer was prepared in the following manner, and was used to introduce activated ester groups on the backbone of a polymer.[0216]

6-Aminocaproic acid, 4.00 g (30.5 mmol), was placed in a dry round bottom flask equipped with a drying tube. Methacrylic anhydride, 5.16 g (33.5 mmol), was then added and the mixture was stirred at room temperature for four (4) hours. The resulting thick oil was triturated three times with hexane and the remaining oil was dissolved in chloroform, followed by drying over sodium sulfate.[0217]

After filtration and evaporation, a portion of the product was purified by silica gel flash chromatography using a 10% methanol in chloroform solvent system. The appropriate fractions were combined, 1 mg of phenothiazine was added, and the solvent was removed under reduced pressure. Analysis on an NMR spectrometer was consistent with the desired product:[0218]¹H NMR (CDCl₃) carboxylic acid proton 7.80-8.20 (b, 1H), amide proton 5.80-6.25 (b, 1H), vinyl protons 5.20 and 5.50 (m, 2H), methylene adjacent to nitrogen 3.00-3.45 (m, 2H), methylene adjacent to carbonyl 2.30 (t, 2H), methyl group 1.95 (m, 3H), and remaining methylenes 1.10-1.90 (m, 6H).

6-Methacrylamidohexanoic acid, 3.03 g (15.2 mmol), was dissolved in 30 ml of dry chloroform, followed by the addition of 1.92 g (16.7 mmol) of N-hydroxysuccinimde and 6.26 g (30.4 mmol) of 1,3-dicyclohexylcarbodiimide. The reaction was stirred under a dry atmosphere overnight at room temperature. The solid was then removed by filtration and a portion was purified by silica gel flash chromatography. Non-polar impurities were removed using a chloroform solvent, followed by elution of the desired product using a 10% tetrahydrofuran in chloroform solvent. The appropriate fractions were pooled, 0.2 mg of phenothiazine were added, and the solvent was evaporated under reduced pressure. This product, containing small amounts of 1,3-dicyclohexylurea as an impurity, was used without further purification. Analysis on an NMR spectrometer was consistent with the desired product:[0219]¹H NMR (CDCl₃) amide proton 5.60-6.10 (b, 1H), vinyl protons 5.20 and 5.50 (m, 2H), methylene adjacent to nitrogen 3.05-3.40 (m, 2H), succinimidyl protons 2.80 (s, 4H), methylene adjacent to carbonyl 2.55 (t, 2H), methyl 1.90 (m, 3H), and remaining methylenes 1.10-1.90 (m, 6H). The final compound was stored for use in the synthesis of photoactivatable polymers as described herein.

Example 6Preparation of Copolymer of Acrylamide, BBA-APMA, and MAL-EAC-NOS (Photo PA-PolyNOS)

A photoactivatable copolymer of the present invention was prepared in the following manner. Acrylamide, 4.298 g (60.5 mmol), was dissolved in 57.8 ml of tetrahydrofuran, followed by 0.219 g (0.63 mmol) of Compound III, prepared according to the general method described in Example 3, 0.483 g (1.57 mmol) of Compound IV, prepared according to the general method described in Example 4, 0.058 ml (0.39 mmol) of N,N,N′,N′-tetramethylethylenediamine (TEMED), and 0.154 g (0.94 mmol) of 2,2′-azobisisobutyronitrile (AIBN). The solution was deoxygenated with a helium sparge for 3 minutes, followed by an argon sparge for an additional 3 minutes. The sealed vessel was then heated overnight at 60° C. to complete the polymerization. The solid product was isolated by filtration and the filter cake was rinsed thoroughly with THF and CHCl[0220]₃. The product was dried in a vacuum oven at 30° C. to give 5.34 g of a white solid. NMR analysis (DMSO-d₆) confirmed the presence of the N-oxysuccinimide (NOS) group at 2.75 ppm and the photogroup load was determined to be 0.118 mmol BBA/g of polymer. The MAL-EAC-NOS composed 2.5 mole % of the polymerizable monomers in this reaction to give Compound VI-A.

The above procedure was used to prepare a polymer having 5 mole % Compound IV. Acrylamide, 3.849 g (54.1 mmol), was dissolved in 52.9 ml of THF, followed by 0.213 g (0.61 mmol) of Compound III, prepared according to the general method described in Example 3, 0.938 g (3.04 mmol) of Compound IV, prepared according to the general method described in Example 4, 0.053 ml (0.35 mmol) of TEMED and 0.142 g (0.86 mmol) of AIBN. The resulting solid, Compound VI-B, when isolated as described above, gave 4.935 g of product with a photogroup load of 0.101 mmol BBA/g of polymer.[0221]

The above procedure was used to prepare a polymer having 10 mole % of Compound IV. Acrylamide, 3.241 g (45.6 mmol), was dissolved in 46.4 ml of THF, followed by 0.179 g (0.51 mmol) of Compound III, prepared according to the general method described in Example 3, 1.579 g (5.12 mmol) of Compound IV, prepared according to the general method described in example 4, 0.047 ml (0.31 mmol) of TEMED and 0.126 g (0.77 mmol) of AIBN. The resulting solid, Compound VI-C, when isolated as described above, gave 4.758 g of product with a photogroup load of 0.098 mmol BBA/g of polymer.[0222]

Example 7Synthesis of Multi-Ligand Conjugate

A multi-ligand conjugate is prepared as follows. A 200 μl aliquot of polystyrene microbeads (1 μm diameter) (obtained from Prolabo, Bangs Labs, Carmel, Ind., product number K100), containing 10% by weight microbeads in aqueous suspension, are mixed with a solution of Photo-PA-PolyNOS (prepared as described in Example 6 above) containing 1.0 mg of polymer in 200 μl of deionized water. The solution is mixed for one hour at room temperature.[0224]

After mixing for one hour at room temperature, the suspension is illuminated for two minutes while mixing, using a Dymax lamp (25 mjoule/cm[0225]²as measured at 335 nm with a 10 nm band pass filter on an International Light radiometer). The microbeads are then washed twice with 2 ml of deionized water by centrifugation at 15,000 rpm for 15 minutes and resuspension in phosphate buffer solution (PBS, pH 7.5).

The washed beads are then incubated with a solution containing a mixture of two oligonucleotide sequences, each modified to contain a primary amine at the 3′ end, and biocytin (10 μM) in 0.05M phosphate buffer, pH 7.5. The first oligonucleotide sequence is the complement to the known sequence of a specific address oligonucleotide provided on a master array. The first oligonucleotide is provided as a 20-mer, at 5 μM. The first oligonucleotide is synthesized by first determining the sequence of a specific address oligonucleotide to be used in the invention. The complement to the address oligonucleotide is then determined using Watson-Crick base pairing rules. Once the complementary sequence is determined, the first oligonucleotide is synthesized using standard nucleic acid synthesis techniques, for example, solid phase synthesis.[0226]

The second oligonucleotide sequence is an analytical sequence that is complementary to a known sequence of a target nucleic acid suspected to be present in a test sample. The second oligonucleotide is provided as a 30 mer, at 10 μM. The second oligonucleotide is synthesized by first determining the sequence of a specific target ligand to be detected using the probe array of the invention. Tthe complement to the target ligand is determined using Watson-Crick base pairing rules. Once the complementary sequence is determined, the second oligonucleotide is synthesized using standard nucleic acid synthesis techniques.[0227]

The microbead suspension is incubated with the oligonucleotides overnight at room temperature. After overnight incubation, the beads are washed twice with 1 ml aliquots of phosphate buffer solution (PBS, pH 7.5), then stored at 4° C. until used. A series of multi-ligand conjugates, each with unique address and analytical sequences is made in this manner for replicating probe arrays.[0228]

Example 8Synthesis of a Spaced Epoxide Monomer (Compound VII)

To three ml of chloroform was added isocyanatoethylmethacrylate (1.0 ml, 7.04 mmol), glycidol (0.50 ml, 7.51 mmol) and triethylamine (50 μl, 0.27 mmol). The reaction was stirred at room temperature overnight. The product was purified on a silica gel column and the structure confirmed by NMR. The yield was 293 mg (18% yield).[0229]

Example 9Preparation of Copolymer of Acrylamide, BBA-APMA, and Glycidyl Methacrylate (Photo PA-Polyepoxide) (Compound VIII)

Acrylamide (7.1 g, 99.35 mmol), BBA—APMA (0.414 g, 1.18 mmol) and 2,2′-azobis(2-methylbutyronitrile) (“VAZO 67”, 0.262 g, 1.4 mmol) were dissolved in 108 ml of THF. To this solution was added 2.4 ml of glycidylmethacrylate (17.7 mmol). The solution was sparged with helium for four minutes, then with argon for four minutes, then capped tightly and heated at 61° C. overnight while mixing. The polymer was collected by filtration, then suspended in methanol and mixed, after which it was again collected by filtration and washed with chloroform, then dried in a vacuum oven at 30 ° C.[0230]

Example 10Preparation of Microscope Slides Coated with PolyEpoxide

Soda lime glass microscope slides (Erie Scientific, Portsmouth, N.H.) were silane treated by dipping in a mixture of p-tolyldimethylchlorosilane (T-silane) and N-decyldimethylchlorosilane (D-Silane, United Chemical Technologies, Bristol, Pa.), 1% each in acetone, for 1 minute. After air drying, the slides were cured in an oven at 120° C. for one hour. The slides were then washed with acetone followed by distilled water dipping. The slides were further dried in an oven for 5-10 minutes.[0231]

Compound VIII (Example 9) was sprayed onto the silane treated slides, which were then illuminated using a Dymax lamp (25 mjoule/cm[0232]²as measured at 335 nm with a 10 nm band pass filter on an International Light radiometer) while wet, washed with water, and dried.

Example 11Preparation of Microarrays with Photo-polyepoxide Coated Slides

Oligonucleotides, either aminated or nonaminated, were printed onto slides at 8 μM in 0.15 M phosphate buffer using an X, Y, Z motion controller to position ChipMaker 2 microarray spotting pins (Telechem International). The slides were either coated with photo-PA-polyepoxide as in Example 10, with photo-PA-PolyNOS by the same procedure or with polylysine by published methods (See U.S. Pat. No. 5,087,522, Brown et al., “Methods for Fabricating Microarrays of Biological Samples,” and the references cited therein).[0233]

The printed epoxide and NOS slides were incubated overnight at room temperature and 75% relative humidity. The printed polylysine slides were processed by the published procedure. The slides were scanned to measure the Cy3 fluorescence of immobilized capture oligonucleotides, then hybridized at 41° C. overnight with Cy5-labeled oligonucleotide (1 pmole/slide) that was complementary to the capture oligonucleotides. The slides were scanned with a laser scanner to measure the fluorescence intensities of the hybridized oligonucleotides. Because the amount of capture oligonucleotide immobilized with the amine-silane slides was low, they were not hybridized.[0234]

TABLE 1


Compounds.




COMPOUND I



COMPOUND II



COMPOUND III



COMPOUND IV



COMPOUND V



COMPOUND VI



COMPOUND VII



COMPOUND VIII

(where x=0.1 to 5 mole %, y=2 to 3 mole %,and z=65 to 97.9 mole %)[0235]

While a preferred embodiment of the present invention has been described, it should be understood that various changes, adaptations and modifications may be made therein without departing from the spirit of the invention and the scope of the appended claims.[0236]